Defining the structure of the substrate-free state of the DnaK molecular chaperone

Abstract

Members of the Hsp70 (heat-shock protein of 70 kDa) family of molecular chaperones bind to exposed hydrophobic stretches on substrate proteins in order to dissociate molecular complexes and prevent aggregation in the cell. Substrate affinity for the C-terminal domain of the Hsp70 is regulated by ATP binding to the N-terminal domain utilizing an allosteric mechanism. Our multi-dimensional NMR studies of a substrate-binding domain fragment (amino acids 387-552) from an Escherichia coli Hsp70, DnaK(387-552), have uncovered a pH-dependent conformational change, which we propose to be relevant for the full-length protein also. At pH 7, the C-terminus of DnaK(387-552) mimics substrate by binding to its own substrate-binding site, as has been observed previously for truncated Hsp70 constructs. At pH 5, the C-terminus is released from the binding site, such that DnaK is in the substrate-free state 10-20% of the time. We propose that the mechanism for the release of the tail is a loss of affinity for substrate at low pH. The pH-dependent fluorescence changes at a tryptophan residue near the substrate-binding pocket in full-length DnaK lead us to extend these conclusions to the full-length DnaK as well. In the context of the DnaK substrate-binding domain fragment, the release of the C-terminus from the substrate-binding site provides our first glimpse of the empty conformation of an Hsp70 substrate-binding domain containing a portion of the helical subdomain.

Characterization of Two Partially Unfolded Intermediates of the Molecular Chaperone DnaK at Low pH

“…Using the same reagent concentrations as described above, we could not detect any binding of either the fCro peptide or the fp5 peptide to ADP-DnaK at pH 4.5 (data not shown). Weak binding for three different peptides suggests a conformational change in the peptide-binding domain of DnaK; this is in accord with recent results from an NMR experiment conducted at low pH (39). In addition, when a sample of ADP-DnaK was preincubated at pH 4.5 with 10 µM fNR for 15 min, and then the pH of the sample was raised to pH 7, the amount of fNR binding to ADP-DnaK increased substantially (data not shown) and was nearly the same as for samples incubated at pH 7 (Figure 5B).…”

Glutathionylation of the Bacterial Hsp70 Chaperone DnaK Provides a Link between Oxidative Stress and the Heat Shock Response

“…1 B ). Truncation of residues 553–638 of DnaK (Δ553–638DnaK) destroys the SBDα lid structure, causing it to bind to the SBDβ, and thus this mutant mimics substrate-bound DnaK ( 5 , 41 ). We found that Δ553–638DnaK had very low cysteine reactivity ( Fig.…”

Chaperones and chaperone–substrate complexes: Dynamic playgrounds for NMR spectroscopists

“…Extension of this study to a twodomain construct containing both NBD and SBD enabled for the first time analysis of the relative orientation of the two domains on the basis of chemical shifts as well as RDCs, highlighting a relatively rigid overall structure that shows allosteric adaptations due to nucleotide and/or substrate binding which are transmitted via subtle structural changes at the domain interface [123]. In parallel, studies on E. coli DnaK by the Gierasch lab revealed initially pH-driven conformational equilibria within the SBD, and showed by chemical shift perturbation and hydrogen/deuterium exchange that for the two-domain construct there is no interaction in the nucleotide-free state and that only nucleotides drive the allosteric adaptations [149,150].…”